New strains of pseudomonas putida

ABSTRACT

Strains of Pseudomonas putida selected from Pseudomonas putida NCIB 12190 and mutant strains thereof, which mutant strains can be obtained by chemical and physical mutation, allowing the mutated bacteria to grow prior to exposure to benzene or fluorobenzene, and subsequently, after further growth in the presence of benzene or fluorobenzene, selecting those mutant strains which accumulate cis-dihydroxycyclohexadiene or catechol or their fluorinated analogues. The new strains can be used in biochemical processes for the preparation of cis-dihydroxycyclohexadienes and catechols.

This invention relates to new strains of Pseudomonas putida and the useof these strains in selective biochemical processes for the productionof dihydroxycyclohexadienes and catechols from benzene and certainderivatives thereof.

The ability of the organism Pseudomonas putida to metabolise benzene andcertain substituted benzenes to their corresponding catechols andfurther degradation products is known from the work of Gibson et al,Biochemistry, 7(7), 1968, p. 2653; and Biochemistry, 9(7), 1970, p.1631. Thus, the metabolism is believed to follow the following enzymecatalysed reaction sequence: ##STR1##

In accordance with this metabolic pathway, benzene (I; R=H) is convertedby a dioxygenase to cis-1,2-dihydroxycyclohexa-3,5-diene (II; R=H)(sometimes known as "cis-benzene glycol" or "benzene dihydrodiol") whichunder the action of a diol dehydrogenase is converted to catechol (III;R=H) which is enzymatically converted to further degradation products. Arelated pathway, where R is methyl, is believed to occur for toluenemetabolism using Pseudomonas putida (Gibson et al, Biochemistry, 9(7),1970, p. 1627).

While compounds of formulae (II) and (III) would be useful products orintermediates to obtain by a biochemical process, it has been founddifficult to control the reaction to give sufficient yield of thedesired product without contamination by the other metabolic products.Attempts to produce, for example, a compound of formula (III) from bothwild type and mutant strains of P.putida have relied on the need toinduce the required enzymes for the conversion reactions using benzeneor toluene as carbon source. Thus Gibson et al (Biochemistry 7(11),1978, p. 3795) used toluene as carbon source when carrying outinvestigations on the ability of P.putida to oxidise halogenatedbenzenes, while Taylor (European Pat. No. 0076606) likewise employedtoluene to induce the required enzymes in his preparation of compoundsof formula (II) from certain mutant strains of P.putida. Such inductionprocedures are disadvantageous as the introduction of another carbonsource for induction purposes contaminates the reaction mixture andnecessitates complex separation problems before a pure product can beobtained.

Taylor (European Pat. No. 0076606) was able to obtain certaindihydroxycyclohexadienes without induction by further mutating a certainmutant strain of Pseudomonas putida. The separation of these secondmutants was made by a selection procedure which involved spraying withcatechol and selecting colonies which gave a colour reaction indicativeof the conversion of catechol into 2-hydroxymuconic semialdehyde, i.e. aring fission between positions 2 and 3 of the catechol ring, whereas analternative metabolic pathway involves fission between the 1 and 2positions of the catechol ring to give a muconic acid.

We have now found a wild type strain of Pseudomonas putida which isconstitutive of the necessary enzymes in the preparation of certaincatechols of formula (III). In other words, we have found a strain inwhich the necessary enzymes do not have to be induced. This strain isthat deposited with effect from 6th Dec. 1985 with the NationalCollection of Industrial Bacteria, Torry Research Station, Aberdeen,Scotland and assigned the numerical designation NCIB 12190 and referredto herein as "P.putida" NCIB 12190".

P.ptuida NCIB 12190 was isolated from a soil sample taken from groundwithin the Shell Refinery at Pernis, Rotterdam.

Pseudomonas putida NCIB 12190 has been characterised and identified bythe NCIB as follows:

Tests were at 25° C. and growth was on LAB M Nutrient Agar unlessotherwise stated.

Cell Morphology

After growth for 24 hours at 30° C. on succinate agar and transfer toNutrient broth+0.75% w agar, by phase contrast at×630 magnification thecells are small short rods or cocci in clusters.

Gram Negative

Spores

Motility+

Colonial Morphology

After 48 hours growth, colonies are round, regular, entire, smooth,opaque, low convex, off-white and less than 1 mm in diameter. Growth onGlucose Peptone Water Sugars

37° C.+

47° C.-

Catalase+

Oxidase, Kovacs+

O-F glucose Oxidative

"O-F glucose" was performed using the oxidation-fermentation medium ofHayward and Hodgkiss, J. Gen. Microbiol, 26. (1961), pp. 133-140,supplemented with 1% w filter-sterilised D-glucose. A tube sample wasinoculated and incubated for 14 days.

Pseudomonas putida NCIB 12190 can conveniently be stored on nutrientagar slopes at 4° C., or as a freeze-dried material.

The UV mutant of Pseudomonas putida NCIB 12190 described in Example 1had the same characteristics as those described above, with theexception of motility-negative.

We have further found that mutants can be obtained directly fromPseudomonas putida NCIB 12190, which mutant strains can be used tocatalyse the reactions of the above described reaction sequence withoutthe need for enzyme induction. Furthermore, we have found that certainmutants catalyse preferentially the conversion of (I) to (II), thusmaximizing the yield of the cis-dihydroxycyclohexadiene and suppressingthe later reactions in the sequence, while other mutants catalyse theconversion from (I) through to (III), but not the conversion to furtherdegradation products. Therefore the reaction can be tailored by use ofthe appropriate mutant to give the desired product of formula (II) or(III).

According to this invention we provide strains of Pseudomonas putidaselected from Pseudomonas putida NCIB 12190 and mutant strains thereof,which mutant strains are capable of accumulatingcis-dihydroxycyclohexadiene or catechol or their fluorinated analogueswhen cultured in the presence of benzene or fluorobenzene and areobtainable by a selection procedure which comprises mutating Pseudomonasputida NCIB 12190 by chemical or physical means, allowing the mutatedbacteria to grow in the presence of a carbon source, exposing the grownbacteria to benzene or fluorobenzene and selecting those mutant strainswhich have accumulated cis-dihydroxycyclohexadiene or catechol or theirfluorinated analogues.

The mutation may be carried out by chemical means, employing, forexample, as mutating agent N-methyl-N'-nitro-N-nitrosoguanidine(referred to hereinafter as ("NTG")). Other mutating agents may beemployed, such as 2-aminopurine or 5-bromouracil. The mutation may alsobe carried out by physical means for example by ultraviolet irradiationor using other ionizing radiation methods.

Examples of preferred carbon sources are succinic acid and fumaric acid,suitably in the form of salts such as disodium succinate and disodiumfumarate. Other suitable carbon sources include sucrose, galactose,lactose, citrates, fructose and glycerol.

An alternative preferred carbon source has been shown to be molasses,both in the form of sugar cane molasses, commercially available as"black strap molasses", and in the form of beet molasses.

The nutrient medium may be any suitable medium for the growth of themutant strains, an example being a minimal salts medium containingsodium succinate.

Preferred strains of Pseudomonas putida are mutant strains. Particularlypreferred mutant strains ar those designated NTG-mutant F and UV-mutantsA and B, described hereinafter.

The invention further includes the use of the new mutant strains inbiochemical processes for the production of cis-dihydroxycyclohexadienesand catechols.

Therefore according to a further aspect of this invention we provide abiochemical process for the preparation of a compound of formula (II);##STR2## from a compound of formula (I) wherein R is hydrogen orfluorine, comprising providing a culture of a mutant strain of P putidawhich accumulates cis-dihydroxycyclohexadiene or its fluorinatedanalogue in the selection procedure defined above, supplying a compoundof formula (I) to the culture in a suitable medium and subsequentlyrecovering a compound of formula (II) therefrom.

According to an alternatiave aspect of this invention we provide abiochemical process for the preparation of a compound of formula (III)##STR3## from a compound of formula (I ##STR4## ) wherein R is hydrogen,or fluorine, comprising providing a culture of P.putida NCIB 12190 or amutant strain thereof which accumulates catechol or 3-fluorocatechol inthe selection procedure defined above, supplying a compound of formula(I) to the culture in a suitable medium and subsequently recovering acompound of formula (III) therefrom.

A preferred medium for the production of the compounds of formulae (II)and (III) comprises sodium succinate as carbon source, for example incombination with ammonium sulphate. Other suitable carbon sourcesinclude fumarates, frutose, glycerol, sucrose, galactose, lactose andcitrates. A further preferred carbon source is molasses, in the form ofsugar cane molasses or beet molasses. The use of molasses as carbonsource in the biochemical production of cis-dihydroxycyclohexadienes andcatechols from the corresponding benzenes is the subject of ourco-pending application Ser. No. 068,491, filed July 19, 1987.

If desired a protein synthesis inhibitor such as chloramphenicol may beincluded in the process for preparing compounds (II) or (III) to enhanceaccumulation of the desired products.

The desired product compound may be recovered from the resultingfermentation broth by any suitable means such as absorption ontogranulated charcoal followed by stripping with a suitable solvent, orsolvent extraction.

While the starting material of formula (I) may be benzene, a preferredstarting material is fluorobenzene, giving as products3-fluoro-cis-1,2-dihydroxycyclohexa-3,5-diene (II : R=F) and3-fluorocatechol (III; R=F). The former can be readily used as anintermediate in its own right or can be converted by chemical means to3-fluorocatechol, 2-fluorophenol or 3-fluorophenol. 3-Fluorocatechol isdifficult to prepare by purely chemical means and is expensive. It is auseful intermediate in the production of, for example, pharmaceuticalsand organo-fluorine agrochemicals.

Examples of preferred mutants for the preparation of 3-fluorocatecholare those described hereinafter and designated UV-mutant A, andUV-mutant B which can be obtained by UV mutagenesis followed by exposureof the mutant organism to fluorobenzene.

The invention will now be described further by way of example.

Colorimetric determination of cis-dihydroxycyclohexadienes and catechols

The methods of Friestad et al., Analytical Chemistry 41, 1969, pp.1750-1754 was used. Solutions to be tested for catechols were mixed withan equal volume of 3-methyl-2-benzothiazolinone hydrazone hydrochloride(0.05% w/v in water) and an equal volume of ceric ammonium sulphate(0.2% w/v in 0.4% w/v sulphuric acid). Optionally, a borate-NaOH-EDTAbuffer was added after a few minutes, as described by Friestad et al.The intensities of the colours which developed were compared visuallyor, alternatively, were measured spectrophotometrically at 520 nm.Cis-dihydroxycylohexadienes do not produce colours under theseconditions. Therefore a second sample of each test solution wasacidified with sulphuric or hydrochloric acid before the colour test, toconvert the dihydroxycyclohexadienes quantitatively to phenols. Thedifference in colour intensity given by the acidified and unacidifiedsamples is indicative of the content of dihydroxycyclohexadienes.

Media Used

The compositions of two of the media used in the following examples aregiven below in Table 1.

ASM--minimal salts medium

NFSM--nitrogen-free salts medium

                  TABLE 1                                                         ______________________________________                                                       amounts per liter                                                             ASM           NFSM                                             ______________________________________                                        Na.sub.2 HPO.sub.4                                                                             0.866   g       7     g                                      KH.sub.2 PO.sub.4                                                                              0.531   g       3     g                                      NH.sub.4 Cl      0.535   g       --                                           K.sub.2 SO.sub.4 0.174   g       0.174 g                                      MgSO.sub.4.7H.sub.2 O                                                                          0.037   g       0.037 g                                      CaCl.sub.2.2H.sub.2 O                                                                          0.00735 g       0.00735                                                                             g                                      TK3 (T/E) (see below)                                                                          1.0     ml      1.0   ml                                     FeSO.sub.4.7H.sub.2 O(0.1M)                                                                    0.2     ml      0.2   ml                                                    pH 6.8                                                         ______________________________________                                        Composition of TK/3 Trace Element Solution:                                   This contained per liter the following components:                            ZnSO.sub.4.7H.sub.2 O (0.288 g); MnSO.sub.4.4H.sub.2 O (0.224 g); H.sub.3     BO.sub.3 (0.0618 g);                                                          CuSO.sub.4.5H.sub.2 O (0.1248 g); Na.sub.2 MoO.sub.4.2H.sub.2 O (0.0484       g); CoCl.sub.2.6H.sub.2 O                                                     (0.0476 g); KI (0.083 g); 1M H.sub.2 SO.sub.4 (1 ml).                         Further media used were:                                                      dYT medium      16 g Bacto tryptone                                                           10 g Bacto yeast extract                                                      5 g NaCl/1                                                    Yeast extract                                                                 medium (YEM)    10 g/l disodium succinate .6H.sub.2 O                                         2 g/l (NH.sub.4 ).sub.2 SO.sub.4                                              3 g/l yeast extract (Difco)                                                   0.4 g/l MgSO.sub.4.7H.sub.2 O                                                 0.04 g/l Bacto-peptone in                                                     25 mM potassium phosphate                                                     buffer, final pH 7.0.                                         ______________________________________                                    

Thin layer chromatography (TLC)

Aqueous samples (5 μl) were run on Merck Kieselgel 60 F254 platesdeveloped with 90:10:1 (v/v) n-propanol-water-formic acid.Dihydroxycyclohexadiene content was visualised under short wave uv lightand catechol was detected by spraying with2,6-dichloroquinone-4-chloroimide (2% in ethanol).

Gas chromatography (GC).

Aqueous samples (0.5 μl) were chromatographed on a Hewlett Packard25-metre high capacity flexible silica capillary column coated with5%-phenylmethylsilicone, using helium as carrier gas. The column washeld at 130° C., and eluted compounds were detected by a flameionisation detector.

EXAMPLE 1

The preparation of NTG mutants of P.putida NCIB

12190

Pseudomonas putida NCIB 12190 was grown overnight at 30° C. in dYTmedium. The culture was subcultured 1/20 into 10 ml fresh dYT andincubated for a further 4 hours. The bacteria were harvested bycentrifugation, washed in NFSM and again harvested by centrifugationbefore resuspension in NFSM (3 ml) and adjustment of the OD 600 nm to3.0.

A 1 ml aliquot in an "Eppendorf" tube was incubated at 30° C. for 15mins and then NTG (N-methyl-N'-nitro-N-nitrosoguanidine-5 mg/ml indimethyl sulphoxide) added to give 50 μg/ml. The tube contents werebriefly mixed and incubated at 30° C. for 15 mins. Mutation was stoppedby chilling in ice, centrifugation and washing (3 times in saline-0.85%NaCl). The bacteria were diluted in saline and 0.1 ml aliquots platedout onto 100 ASM plates each including 0.05% sodium succinate. Theplates were incubated at 30° for 48 hours.

After incubation, small "micro" colonies were visible on the plates, thelevel of succinate having been only sufficient for a small amount ofgrowth. The plates were then exposed to benzene vapour for 24 h at 30°C. resulting in a wide variety of colony sizes visible on the plates. Inan initial selection procedure, small colonies on the plates (which aresmall because of their inability to grow on benzene ) (20-30/plate) werepicked using sterile cocktail sticks into 96 well microtitre plates(Titertek) containing 50 μl ASM+0.5% sodium succinate per well. Theplates were incubated overnight at 30° C. in a sealed box to preventevaporation and the microtitre plates replicated onto square (120 mm)plates of commercial nutrient agar (oxoid) (to provide controls) ussinga 96 prong replicating device. The agar plates were incubated overnightand the microtitre plates were exposed to benzene vapour for 3 hours.Subsequent assay was by the colorimetric method described above. Thosemutants developing the most intense colour were selected for furthertesting for the accumulation of catechol an cis-dihydroxycyclohexadieneand identification of the best mutants for production of each. Theselected best mutants wre tested using shake flask cultures and thecolorimetric determination again carried out. Controls were run using aculture of wild type P.putida NCIB 12190 to which chloramphenicol hasbeen added as a protein synthesis inhibitor, although no chloramphenicalwas added to the mutant strains. Confirmatory HPLC analyses of some ofthe supernatants were carried out.

From each of six independent starting cultures of P.putida NCIB 12190mutated with NTG, 100 plates were innoculated giving approximately100,000 colonies, 13,500 of which were picked into microtitre plates and435 initially selected for colour testing. 90 of these were furtherinvestigated after colour testing and TLC analysis and shake flaskexperiments carried out on 14 of the mutants.

The cultures were grown overnight in ASM plus 0.5% sodium succinate,harvested by centrifugation, washed and resuspended in NFSM. Thecultures were exposed to benzene and the supernatants assayed forcatechol or catechol plus cis-dihydroxycyclo- hexadiene. For controlruns, wild type P.putida NCIB 12190 was employed with chloramphenicol (1mg/ml) added to the NFSM medium.

Catechol producing mutants

Several mutants produced amounts of catechol at least comparable tothose produced by a control using wild type P-putida NCIB 12190 pluschloramphenicol, even though the mutants did not need the presence ofchloramphenicol.

These mutants are hereinafter referred to as "NTG-mutants A, B, C andD". The results for mutants B, C and D are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                         OD 520 nm.                                                   Strain             after 3 hrs                                                                             after 7 hrs                                      ______________________________________                                        Mutant B           0.25      0.49                                             Mutant C           0.21      0.47                                             Mutant D           0.34      0.58                                             NCIB 12190 + chloramphenicol                                                                     0.23      0.47                                             ______________________________________                                    

Cis-dihydroxycyclohexadiene producing mutants

Two mutants were unable to produce catechol but accumulatedcis-dihydroxycyclohexadiene. These mutants are hereinafter referred toas "NTG-mutants E and F". The results for mutants E and F are shown inTable 3.

                  TABLE 3                                                         ______________________________________                                        OD 520 nm                                                                                        Catechol plus                                              Catechol           cis-dihydroxycyclohexadiene                                Strain after 21/2 hrs                                                                          after 7 hrs                                                                             after 21/2 hrs                                                                         after 7 hrs                               ______________________________________                                        Mutant E                                                                             0.02      0.02      0.17     0.24                                      Mutant F                                                                             0.04      0.06      0.27     0.54                                      ______________________________________                                    

The advantageous effect on diene production of the addition of a proteinsynthesis inhibitor (chloramphenicol-1mg/ml) to mutants E and F is shownin Table 4

                  TABLE 4                                                         ______________________________________                                                         OD 520 nm                                                                     Catechol plus                                                                 cis-dihydroxycyclohexadiene                                  Strain             after 21/2 hr                                                                            after 7 hr                                      ______________________________________                                        Mutant E           0.17       0.24                                            Mutant E plus chloramphenicol                                                                    0.49       0.75                                            Mutant F           0.27       0.54                                            Mutant F plus chloramphenicol                                                                    0.34       0.85                                            ______________________________________                                    

EXAMPLE 2 The production of NTG mutants of P.putida

Example 1 was repeated as far as the initial selection procedure and 450candidate organisms from the initial selection procedure were receivedas purified cultures on agar plates and inoculated into 1.5 ml of an ASMmedium (supplemented with a trace element mixture, Fe²⁺ -20 μm andsodium succinate - 5 g/l). The cultures were grown overnight in slopingrotating test tubes at 30° C., centrifuged, the supernatants discardedand the cells resuspended in 0.25 ml supplemented ASM in test tubesarranged nearly horizontally in a tank of benzene vapor on a rockingplatform at room temperature.

After a certain benzene exposure time (usually 3 and/or 5 to 6 hours)two 0.5 ml samples of each culture were withdrawn for colour testing bythe colorimetric method described above. To one of these were added 10μl of 5M HCl to convert any cis-dihydroxycyclohexadiene present intophenol. 5 μl samples were also taken for TLC analysis. After the benzeneexposure, the cultures were left in air and a final TLC sample takennext day.

Two cultures of wild type P.putida NCIB 12190 were included in eachbatch tested for comparison purposes, to one of which chloramphenicol(0.1 mg/ml) had been added before exposure to benzene. Colours and TLCposts were compared and scored on an eight-point scale from--to +++. Theresults are given in Table 5. Reproducability was found to be good.

                                      TABLE 5                                     __________________________________________________________________________                3 hr benzene exposure                                                                            5-6 hr benzene exposure                                    Colour Test                                                                             TLC      Colour Test                                                                             TLC (next day)                       Mutant      + acid                                                                             no acid                                                                            Diene                                                                             Catechol                                                                           + acid                                                                             no acid                                                                            Diene                                                                             Catechol                         __________________________________________________________________________    CONTROLS                                                                      NCIB 12190  +(+) +(+) -   +(+) ++   ++   -   +                                NCIB 12190 +                                                                  chloramphenicol                                                                           +++  +++  -   +++  +++  +++  -   +++                              CIS-DIHYDROXY-                                                                CYCLOHEXADIENE                                                                PRODUCERS                                                                     NTG mutant E                                                                              (+)                -(+) -    (+) -                                NTG mutant G                                                                              (+)  -(+) (+) -    (+)  -(+) -   -                                NTG mutant H                                                                              +    -(+) (+) -    +    -(+) (+) -                                NTG mutant F                                                                              +(+ )                                                                              -(+) +   -    +    -(+) +   -                                NTG mutant I                                                                              -(+) -    -   -    -(+) -    -   -                                NTG mutant J                                                                              (+)  -(+) -   -    (+)  -(+) -   -                                NTG mutant K                                                                              ++                 ++   (+)  -   (+)                              NTG mutant L                                                                              -(+) -    -   -    -(+) -    -   -                                NTG mutant M                                                                              (+)  -    -   -(+) +    (+)  -   -                                CATECHOL                                                                      PRODUCERS                                                                     NTG mutant C                                                                              ++                 ++   +++  -   +++                              NTG mutant N                                                                              +++  +++  -   +++  ++(+)                                                                              ++(+)                                                                              -   +++                              NTG mutant O                                                                              +++  ++(-)                                                                              -   +++  ++(+)                                                                              ++(+)                                                                              -   +++                              NTG mutant P                                                                              +++  ++(-)                                                                              -   ++(+)                                                                              ++(+)                                                                              ++(+)                                                                              -   +++                              NTG mutant Q                                                                              ++(+)                                                                              ++(-)                                                                              -   ++(+)                                                                              ++(+)                                                                              ++(+)                                                                              -   ++(+)                            NTG mutant A                                                                              ++   ++   -   +++  ++   ++   -   +++                              NTG mutant R                                                                              +++  +++  -   +++  +++  +++  -   ++(+)                            NTG mutant S                   ++   ++(+)                                                                              -   ++(+)                            NTG mutant R                   ++   ++   -   ++(+)                            NTG mutant U                   ++   ++   -   ++(+)                            NTG mutant V                   ++   ++(+)                                                                              -   + ++                             __________________________________________________________________________

EXAMPLE 4 The use of NTG mutant F in the production of3-fluoro-cis-1,2-dihydroxycyclohexa-3,5-diene using succinate as carbonsource.

8 Liters of YEM were inoculated in a stirred fermenter with a 20 hourshake flask culture of NTG Mutant F grown on the same medium (50 ml).The organism was grown at aerating conditions of 500 rpm and 500 ml.air/min. for 19 hours with a continuous feed of concentrated nutrient(320 g of disodium succinate adn 64 g of ammonium sulphate in 1 liter of0.025M potassium phosphate buffer pH 7.2) at 40 ml/hour. Oxygen transferwas increased by increasing the stirrer speed to 550 rpm and theaeration rate to 700 ml air/min for 30 min and then set to conditions of500 rpm; 600 ml air/min; equilibrium oxygen tension 30% air saturatedinitially).

Fluorobenzene was metered by a pump (Gilson Model 302) at 50 μl/min(100-125 mg/l equilibrium concentration in the reaction). Prior toreaction, the optical density as determined as 3.11 corresponding to adry cell weight of 3.9 g/l. Production of3-fluoro-cis-1,2-dihydroxycylohexa-3,5-diene (II; R =F) was monitored bygas chromatography and the concentration reaches 2.8 g/l after 24 hr.

The product was absorbed on granulated charcoal and recovered from thecharcoal by extraction in a Soxhlet apparatus with a mixture of diethylether and methanol (4:1 v/v). Evaporation of the solvent left a solidwhich was recrystallised from a mixture of diethyl ether and pentane togive colourless needles, m.p. 73°-74° C. (decomp.).

UV Spectrum (H₂ O): λ max 259 nm (ε 3150).

Circular dichroism) (H₂ O: λ max 255 nm (Δε - 1.9).

Mass spectrum m/z 130 (15%,M⁺), 112 (65%), 84 (100%).

¹ H-NMR spectrum (CDCl₃): δ 5.88 (1H, mult, J=10, 6.5, 6, 2Hz; 5-C-H),5.71 (1H, dd, J =10, 3 Hz; 6-C-H), 5.60 (1H, dd, J=11, 6.5 Hz; 4-C-H),4.51 (1H, br.mult, J=6, 3, 2 Hz; 1-C-H), 4.29 (1H, tr, J =6, 6 Hz;2-C-H), 2.40 (2H, br.s, O-H) p.p.m.

EXAMPLE 5 The use of NTG mutant F in the production of3-fluoro-cis-1,2-dihydroxycyclohexa-3,5-diene using molasses as carbonsource

A medium of 25 mM phosphate buffer (pH 7.0; 8 l) containing canemolasses (240g), yeast extract (24g), ammonium sulphate (16 g),magnesium sulphate heptahydrate (3.2g) and bactopeptone (0.32 g) wasinoculated with a shake flask culture of NTG mutant F and air was passedat a rate of 500 ml/min into the stirred mixture (500 rpm) over a periodof 20 hours. During the latter 16 hours of growth, the fermenter was fedwith a solution of cane molasses (500 g/l) and ammonium sulphate (50g/l) in 25 mM phophate buffer (pH 7.0) at a rate of 40 ml/hour. The pHof the broth was maintained at about 7.0 by addition via a monitoringsystem of 10% aqueous sodium hydroxide.

After 20 hours incubation, 5 litres of the broth were withdrawn and theremainder diluted with 25 mM phosphate buffer (pH 7.0; 51) containingammonium sulphate (5g). The mixture was aerated under conditions of 750ml/min; 600 rpm and fluorobenzene (0.2 ml) added. A solution of canemolasses (250 g/l) and ammonium sulphate (25 g/l) in 25 mM phosphatebuffer (pH 7.0) was added at a rate of 40 ml/hour and fluorobenzene wasadded at a rate of 50 μl/min. Air feed and stirring were adjusted tomaintain a positive (about 30%) oxygen tension. After 5 hours, the airfeed was raised to 800 ml/min and maintained for a further 15 hours.

The product reached a level of greater than 9 g/l and was isolated fromthe broth by absorption onto charcoal. Elution of the charcoal withether/methanol gave product3-fluoro-cis-1,2-dihydroxycyclohexa-3,5-diene (II; R=F; 46g), identicalwith the product of Example 4.

EXAMPLE 6 The preparation of UV mutants of P-putida.

Aliquots of 1 ml of a suspension of P.putida NCIB 12190 in phosphatebuffer pH7 were spread onto nutrient agar plates. The plates wereirradiated using a chromatolux u.v. lamp for 5 to 30 minutes. The plateswere incubated at 30° C. overnight and then placed in an atmosphere offluorobenzene at 30° C. and incubated for a further 24 hours. 34surviving colonies were purifed and tested for their ability toaccumulate fluorocatechol in shake flask experiments. Fluorocatechol wasassayed by gas chromatography. In comparative experiments, one mutantstrain, hereinafter referred to as as UV-mutant A, accumulated 0.68 g/lfluorocatechol in 3-4 hr, and a second mutant strain, designatedUV-mutant B, accumulated 0.56 g/l in 3-4 hr. Under the same conditionsthe wild strain NCIB 12190 accumulated 0.41 g/l

EXAMPLE 7 The use of UV mutant A in the production of 3-fluorocatechol

8 Liters of YEM in a fermenter were inoculated with 30 ml of a 16 hourshake culture of UV-mutant A grown at 30° C. on the same medium. Theorganism was grown under conditions of oxygen limitation (200 rpm, 350ml air/min) for 17 hours, during which time a zero reading was recordedon the oxygen electrode. After 17 hours the aeration rate was increasedto 400 rpm; 450 ml air/min to stimulate growth and maintained for 1 hourto provide an actively growing culture of dry weight 0.6 g/l (viablecell count 1.9×10⁹ /ml). Fluorobenzene (10 ml) was added and aerationdecreased to 320 rpm; 150 ml air/min. The air was saturated withfluorobenzene by passage through a bubbler containing that compound,maintaining an equilibrium concentration of fluorobenzene of about 300mg/l in the reaction mixture. 3-Fluorocatechol production was monitoredby GLC and estimated to have reached 0.45 g/l after 6 hours and 0.62 g/lafter 24 hours. The pH was controlled throughout at 7.2 by automaticaddition of H₂ SO₄.

3-Fluorocatechol (III); R =F) was isolated from the culture broth byabsorption on charcoal and recovered from the charcoal by solventextraction, as described in Example 4. After purification by sublimationand recrystallisation from toluene, the product formed colourlessplatelets, m.p. 75.5°-72° C.

UV spectrum (H₂ O): max 267 nm (1060).

Mass spectrum: m/z 128 (100%, M⁺), 80 (55%), 52 (70%).

¹ H-NMR spectrum (CDCl₃): 6.8-6.65 (3H, mult; Ar-H), 5.72 (1H, br.s;O-H), 5.40 (1H, br.s; O-H) p.p.m.

EXAMPLE 8 The use of Pseudomonas putida NCIB 12190 in the production ofcatechol using succinate as carbon source

P.putida NCIB 12190 was grown overnight at 30° C. in ASM using as carbonsource disodium succinate 0.6H₂ O (0.5%). After growth the bacteria wereharvested by centrifugation, washed and resuspended in NFSM.

The washed and resuspended bacteria were placed into a 250 ml centrewell flask. A few drops of benzene were added to the centre well and theflask sealed with "Nescofilm". The flask was incubated without shakingat 30° C.

Samples were taken at time intervals and following each sampling theflask was freshly sealed. Samples of 0.5 ml were withdrawn and thebacteria were removed by centrifugation for 1 min. in an"Eppendorf"bench centrifuge. The presence of catechol was measured bythe colorimetric method described above.

The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                     OD 520 nm                                                        Strain         after 3 hrs                                                                             after 7 hrs                                          ______________________________________                                        NCIB 12190     0.10      0.17                                                 ______________________________________                                    

EXAMPLE 9 The use of Pseudomonas putida NCIB 12190 in the production ofcatechol using succinate as carbon source and chloramphenicol as proteinsynthesis inhibitor

The process of Example 8 was repeated using disodium succinate .6H₂ O ascarbon source. In one run chloramphenicol (1 mg/ml) was added to theNFSM in which the bacteria were resuspended after growth. Nochloramphenicol was added to the comparison run. Catechol formation wasassayed after exposure to benzene as described in Example 8 and theresults are shown in Table 7:

                  TABLE 7                                                         ______________________________________                                                           OD 520 nm                                                  Strain               after 2 hrs                                                                             after 5 hrs                                    ______________________________________                                        NCIB 12190           0.07      0.04                                           NCIB 12190 plus chloramphenicol                                                                    0.08      0.23                                           ______________________________________                                    

It will be seen that, although both cultures metabolise benzeneimmediately, much higher levels of catechol accumulated in thechloramphenicol treated bacteria. Furthermore, there was no evidence ofbreakdown of catechol in the chloramphenicol treated bacteria.Furthermore, there was no evidence of breakdown of catechol in thechloramphenicol treated cells, while in the untreated cells the amountof catechol decreased after reaching a peak at about 3 hours.

EXAMPLE 10 The use of Pseudomonas putida NCIB 12190 in the production ofcatechol using succinate as carbon source - effect of nitrogenlimitation

The process of Example 8 was repeated using disodium succinate .6H₂ O ascarbon source. Four runs were carried out which differed as follows:

Run A The culture was initially grown in ASM and then resuspended inNFSM without chloramphenicol addition.

Run B The culture was initially grown in ASM and then resuspended inNFSM with the addition of chloramphenicol (1 mg/ml).

Run C The culture was initially grown in NFSM including (NH₄)₂ SO₄(0.05%) and then resuspended in NFSM without the addition ofchloramphenicol.

Run D The culture was initially grown in NFSM including (NH₄)₂ SO₄(0.05%) and then resuspended in NFSM with the addition ofchloramphenicol (1 mg/ml).

Runs C and D were thus nitrogen limited cultures containing 10% of thenitrogen present in Runs A and B. Catechol formation was assayed afterexposure to benzene as described in Example 8 and the results are shownin Table 8:

                  TABLE 8                                                         ______________________________________                                                  OD 520 nm                                                           Run         After 31/2 hrs                                                                           After 61/2 hrs                                         ______________________________________                                        A           0.02       0.02                                                   B           0.33       0.61                                                   C           0.30       0.56                                                   D           0.53       1.03                                                   ______________________________________                                    

The results show that the catechol accumulation was substantiallyenhanced in the nitrogen limited cultures (Runs C and D) compared withRuns A and B where there was no attempt at nitrogen limitation. The useof chloramphenicol enhanced catechol accumulation over and above thenitrogen limitation (Run D).

EXAMPLE 11 The use of Pseudomonas putida NCIB 12190 in the production ofcatechol using various substances as carbon source, with and withoutadded chloramphenicol

The process of Example 8 was repeated using as carbon source thesubstances listed in Table 9. Carboxylic acids were added as theirsodium salts. For each experiment the carbon source, its concentrationin the culture medium, and the resulting cell density after growthovernight, estimated from the turbidity of the culture, are given inTable 9. The production of catechol by the resuspended cells, with andwithout added chloramphenicol (1 mg/ml), was estimated colorimetricallyand is shown in Table 9. An increasing number of "+" signs in Table 9indicates increasing turbidity or intensity of colour.

                  TABLE 9                                                         ______________________________________                                                         Catechol Production                                                    Concen-                     With                                              tration  Cell    Without    chloram-                                Carbon source                                                                           g/l      density chloramphenicol                                                                          phenicol                                ______________________________________                                        Succinate 10       +++     ++         +++                                     Fructose  10       ++      ++(+)      +++                                     Sucrose    4       ±    ±       ++                                      Galactose 10       +       +(+)       ++                                      Lactose   10       ±    +          ++                                      Fumarate  10       ++      +(+)       ++(+)                                   Formate   10       ±    ±       +                                       Citrate   10       ++      +          +(+)                                    Ethanol   10       ±    ±       +                                       Glycerol  10       +(+)    +          +++                                     ______________________________________                                    

EXAMPLE 12 The use of Pseudomonas putida NCIB 12190 in the production of3-fluorocatechol using succinate as carbon source

The process of Example 8 was repeated using disodium succinate .6H₂ O ascarbon source but employing fluorobenzene as feedstock for addition tothe resuspended bacteria in centre well flasks. Two runs (Runs A and B)were carried out using fluorobenzene, which differed in that Run A wascarried out without the addition of chloramphenicol, whereas, for Run B,chloramphenicol (1 mg/ml) was added to the NFSM used for resuspension.

For comparison purposes, two runs (Runs C and D) were carried out usingbenzene which differed in that Run C was carried out without theaddition of chloramphenicol, whereas, for Run D, chloramphenicol (1mg/ml) was added to the NFSM used for resuspension.

Fluorocatechol and catechol formation were assayed as described inExample 8 and the results are shown in Table 10:

                  TABLE 10                                                        ______________________________________                                                   OD 520 nm                                                          Run          After 4 hrs                                                                             After 7 hrs                                            ______________________________________                                        A            0.37      0.63                                                   B            0.31      0.57                                                   C            0.31      0.53                                                   D            0.55      0.97                                                   ______________________________________                                    

The results of Runs A and B show that 3-fluorocatechol is formed fromfluorobenzene but that the use of chloramphenicol as protein synthesisinhibitor has little effect and, indeed, in this case is slightlydisadvantageous.

EXAMPLE 13 The isolation of 3-fluorocatechol from culture broths

3-Fluorocatechol was recovered from culture broths such as thoseproduced by scaled up Run A of Example 12 by centrifugation to removecells, followed by continuous extraction with diethyl ether for 18hours. Evaporation of the ether yielded a white solid which was furtherpurified by sublimation (50-60° C., 10 torr). The product had m.p.65°-68° C. Elemental analysis: Found, C 56.7, H 3.5%; Calc. for C₆ H₅FO₂, C 56.3; H 3.9%. The ultraviolet absorption spectrum, mass spectrum,and ¹ H- and ¹⁹ F-nuclear magnetic resonance spectra agreed with thoseobtained for a synthetic reference sample.

We claim:
 1. A biological pure culture of Pseudomonas putida NCIB 12190.